Genes involved in the determination of segmental identity in Drosophila melanogaster have been identified on the basis of dosage-dependent genetic interactions with genes already known to be involved in the process. Of eighteen genes identified by interacting mutations, twelve were not previously known to be involved in the process. Four of the newly-identified genes, the brahma, kismet, oas, and moira genes, were chosen for more extensive genetic and molecular analyses. Cells lacking either kismet or moira gene products in mosaic individuals survive and express homoerotic transformations in some segments of the adult cuticle. Both maternal and zygotic expressions of the brahma gene are required for survival of the early embryo. Lack of functional brahma products at either stage severely disrupts development. DNA from the region of the genome containing the brahma gene has been isolated and two candidates for the brahma transcriptional unit have been identified by insertional mutagenesis. Each of the two candidates for the putative brahma transcriptional unit encodes a single mRNA species present at all developmental stages examined. cDNA clones corresponding to both mRNA molecules have been isolated and sequenced. DNA from the chromosomal region containing the brahma gene has been isolated. An insertion of P element DNA into the kismet gene has been isolated and characterized in order to clone the wild-type osa gene.